Project Summary/Abstract Recent work within our Lab in pretargeted positron emission tomography (PET) imaging experiments will allow us to expand our portfolio of available bioorthogonal Tz radioligands, based on bioorthogonal click chemistry, utilizing a newly developed 18F-labeled tetrazine (Tz)-derived small molecule imaging probe and a trans- cyclooctene (TCO)-modified anti-CA19.9 antibody 5B1. In order to identify new Tz vectors with improved pharmacokinetics, in vivo pretargeted PET imaging of both pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) will be utilized as our pretargeting platforms with the antigen/antibody pairs, CA19.9/5B1 and A33/huA33, respectively. In particular, and central to this proposed Fellowship research program, we believe that by shortening the clearance time of the radioligand we will be able to increase the tumor-to-background ratio at early imaging time points, addressing the urgent demand for novel, safe and powerful diagnostic tools for PDAC and CRC imaging. By focusing on the 18F-radionuclide as a short-lived radionuclide with a physical half-life below 2 hours, we ultimately endeavor to significantly reduce the off-target radiation burden to patients while maintaining high delineation of tumor tissue. As the modular nature of the bioorthogonal inverse-electron demand Diels-Alder reaction enables the utilization of different antibodies, potential preclinical and clinical applications of novel fast-clearing radioligands for click chemistry-based in vivo pretargeted PET imaging are not limited to PDAC and CRC, but may find important applications in other cancer types as well. Using the pretargeting methodology in combination with the CA19.9/5B1 and A33/huA33 targeting platforms, we believe that positive results may have a tremendous impact on the early diagnosis and staging of these types of cancer. As patient survival with PDAC and CRC dramatically increases when malignant tissue and primary tumors are detected early, we believe that are Lab has identified solutions for target-specific pretargeted PET imaging. Utilizing this short-lived radionuclide, which is indicative as a valuable diagnostic tool in the clinic, would increase the likelihood of early tumor diagnosis in PDAC and CRC. As outlined in the current proposal, we believe that our research plan is of great potential clinical impact and the Aims are of scientific merit, and the funding from the F32 fellowship award would greatly benefit the PI in early career stages. Considering that while pretargeted imaging strategies are still within infancy for research outcomes, our preliminary data illustrate that this methodology has the potential to significantly contribute to the advancement of cancer imaging and therapy in the near future.